Project Summary Some of the most effective currently used anti-cancer drugs create interstrand cross-links (ICLs) in cellular DNA. If left unremoved, these ICLs act as absolute blocks to DNA transcription and replication, and trigger cell death. A growing body of evidence shows that tumor cells can acquire resistance to cross-linking drugs through their ability to repair ICLs. During the previous grant period, we studied the effect of alkyl ICLs on DNA structure and characterized ICL processing and repair in mammalian whole cell extracts and cells. Importantly, we discovered a novel ICL unhooking activity in human whole cell extracts whose efficiency correlates with the degree of helix distortion imparted by the cross-link. In addition, we have begun to identify proteins in mammalian cells that are responsible for ICL repair. The goal of this research project is to understand how ICLs are repaired. This knowledge will be used to: (1) design "cryptic" ICLs that can evade repair and (2) identify the proteins responsible for cross-link repair in order that they may be used as therapeutic targets to enhance the effectiveness of cross-linking drugs. This project has four specific aims. Specific Aim 1 will prepare DNA duplexes that have novel, cryptic N4C-propenyl-N4C or N4C-propenyl-N6A ICLs, that are expected to cause minimal perturbation to the B-form DNA helix and thus be less susceptible to repair. A novel nucleoside, N4-(3-porpanalyl)-dC, will be synthesized and tested for its ability to create the propenyl ICLs in normal and tumor cell DNA. Specific Aim 2 will prepare DNA duplexes having a novel Sp/Sp 3,6-dioxaoctane phosphotriester ICL, a cross-link also predicted to be compatible with B-form DNA. 1,8-Bis(nitrosoureido)-3,6- dioxaoctane, 1,8-bis(methylsulfonyl)-3,6-dioxaoctane and 1,8-bis(sulfamyl)-3,6-dioxaoctane cross-linkers will be synthesized and tested for their ability to create phosphotriester ICLs in cellular DNA. Specific Aim 3 will characterize processing of these novel ICLs in human and hamster whole cell extracts and will identify the protein(s) responsible for ICL unhooking. Specific Aim 4 will use a plasmid reporter system to examine ICL repair in human cells and in human tumor cells that are resistant to therapeutic cross-linking drugs. siRNAs that target the unhooking protein(s) identified in Specific Aim 3 and other proteins involved in ICL repair will be used to determine the effect of knocking down these proteins on ICL repair in human tumor cell lines. We will also determine if these proteins are potential therapeutic targets by knocking them down in tumor cells with siRNAs and determining if this sensitizes the cells to cross-linking drugs. The development of cryptic ICLs and identification of proteins responsible for ICL repair is expected to lead to the development of more effective and perhaps selective anti-cancer chemotherapeutic agents.